Treatment or prevention of menopausal symptoms and osteoporosis

ABSTRACT

There is described a method for the treatment or prevention of menopausal symptoms or osteoporosis wherein there is administered to a subject in need of such treatment a therapeutically effective amount of the isoflavone formononetin, or a method for the treatment or prevention of menopausal symptoms wherein there is administered to a subject in need of such treatment a therapeutically effective amount of the isoflavone daidzein, the isoflavone being optionally administered with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients. Therapeutic uses and compositions/foods are also described, comprising daidzein or formononetin optionally in association with one or more pharmaceutically acceptable adjuvants, carriers, food components and/or excipients.

[0001] This invention relates to compositions, therapeutic uses andmethods of treatment or prevention of menopausal symptoms andosteoporosis.

[0002] Menopausal symptoms and osteoporosis are significant scourges inthe female population, generally affecting many women in later life.

[0003] Menopausal symptoms are very well known and are described, forexample, by Greene, J. G. and Cooke, D. J. (1980) British Journal ofPsychiatry Volume 136, 486-491 (incorporated herein by reference). Hotflushes are one of the principal menopausal symptoms which areuncomfortable and irritating. Greene and Cooke have developed a score inorder to measure menopausal symptoms in women. This score is approved bythe US Department of Health and widely used in the medical community.The indicators of menopausal symptoms according to Greene and Cookecomprise hot flushes, sweating at night, heart beating quickly orstrongly, feelings of tension or nervousness, difficulty in sleeping,excitability, attacks of panic, difficulties in concentrating, feelingsof tiredness or lack of energy, unhappiness or depression, cryingspells, irritability, feelings of dizziness or faintness, pressure ortightness in head or body, parts of the body feeling numb or tingling,dry vagina and/or dry mouth, headaches, muscle and joint pains, loss offeeling in hands or feet, breathing difficulties, and loss of interestin sex.

[0004] The peri-menopausal stage of life in women is associated with afall in blood levels of the three major estrogens—estradiol, estrone andestriol—which occurs naturally in women usually between 45-55 years ofage. The primary or acute menopause symptoms which effect menopausalwomen include hot flushes and night sweats. These are associated withoften dramatically increased blood perfusion of the skin producingdiscomfort and sweating.

[0005] The precise mechanism of these symptoms is unknown but generallyis thought to represent disturbance to normal homeostatic mechanismscontrolling vasomotor activity and thermoregulation.

[0006] The fact that treatment and/or prevention with replacementestrogens usually relieves the symptoms (so-called estrogen replacementtherapies) establishes the link between these symptoms and an estrogendeficiency. The menopausal stage of life is associated with a wide rangeof other acute symptoms as described above and these symptoms aregenerally estrogen-responsive.

[0007] Osteoporosis is believed to affect one third to one half of allpost-menopausal women. In the United States it is been reported flatannually 500,000 bone fractures occur as a result of osteoporosis. It isfurther reported that nearly one third of women over 65 will suffer atleast one bone fracture resulting from osteoporotic bone weakening.Increased calcium intake and other approaches are suggested to have someeffect. However, the widespread effects of osteoporosis indicateseffective approaches for prevention/treatment have not yet arisen.

[0008] It has previously been thought that reduction in endogenousestrogen levels which occurs prior to menopause causes or contributes tothe symptoms of menopause, as well as post-menopausal osteoporosis.

[0009] Isoflavones, being plant chemicals which occur largely in membersof Leguminosae, display a range of biological functions which havesuggested they may be useful in treating a host of medical conditions.

[0010] A small subgroup of isoflavones (comprising daidzein, genistein,biochanin, and formononetin and) is distinguished by their ability tobind to estrogen receptors on animal (including human) cells. This isdue to the close similarity of the steric structure of the diphenolicrings of isoflavones with the steroidal ring structure of estrogens suchas estradiol, estrone and estriol. Although having substantially lowerbinding affinity to the receptor compared to steroidal estrogens,estrogenic isoflavones are weakly estrogenic. This group of fiveisoflavones have the most basic diphenolic structure possible incontrast to the relatively more complex structures of other isoflavonoidcompounds. This simplicity of structure and its close proximity in shapeto the steroidal ring structure of estrogenic hormones is believed togrant these compounds their estrogenicity. This group also exhibits arange of biological functions in animal cells which appear to beindependent of the estrogen receptor and these include antioxidant,diuretic, anti-spasmolytic and anti cancer effects. These interestingfunctions with their potential therapeutic benefits has brought thisparticular group of isoflavones to the attention of medical researchersin recent years.

[0011] In the plant, the isoflavones can occur in a variety of forms—(i)in the basic form, (ii) in a malonyl form, and (iii) in an acetyl form;the isoflavones are biologically active in each of these forms. Thenaturally-occurring state for each of these forms is as a glycoside,being bound to a sugar moiety such as glucose to produce a water-solubleform. In this form, the isoflavone has enhanced stability to degradativefactors such as heat, oxidation and ultraviolet irradiation. Thiswater-soluble form also permits transport of the isoflavone both aroundthe plant and intra-cellularly. At the intra-cellular site ofbiochemical function of the isoflavone, an intra-cellular glucosideenzyme cleaves the sugar moiety, leaving the more biologically active,but water-insoluble, aglucone form.

[0012] When ingested in the diet, the isoflavones undergo varyingdegrees of metabolism within the gut, within the gut wall, and withinthe liver before entering the parenteral bloodstream to exert theirbiological effects. The first metabolic process is the hydrolysis of theglucosidic form to the aglucone form. This occurs as a result both oflow pH from gastric acid and of the action of β-glycosidase enzymeactivity within bowel bacteria.

[0013] Some of the aglucone isoflavones are absorbed intact and inpassing through the gut wall are believed to be glucoronated orsulphonated as per steroidal compounds. The bulk of isoflavones arefermented within colonic bacteria. One of the fermentation processes isto demethylate isoflavones (e.g. formononetin gives daidzein andbiochanin gives genistein on demethylation). In another series offermentation steps, daidzein and genistein are converted to a range ofend-products including equol, dehydroequol, O-desmethylangolensin(ODMA), 6-hydroxy-ODMA, 2-dehydro-ODMA, dihydrodaidzein,tetra-hydrodaidzein and dihydrogenistein. The liver is capable offurther demethylation of isoflavones such as formononetin and biochaninto the more basic daidzein and genistein structures. The isoflavones andtheir metabolites and derivatives circulate freely within the body andare excreted primarily in the urine with smaller amounts in the faeces.

[0014] The possibility that dietary estrogenic isoflavones may have sometherapeutic benefit in acute menopausal symptoms was suggested by theobservation that Japanese women who typically have much higher dietarylevels of isoflavones (mostly derived from soya) compared to women inWestern countries have a reportedly lower incidence of acute menopausalsyndrome symptoms such as hot flushes. This has led to somewhatspeculative claims of therapeutic benefit of the isoflavones from thegroup daidzein, formononetin, biochanin and genistein in the treatmentand/or prevention of acute menopausal syndrome symptoms (U.S. Pat. No.5,498,631—Gorbach et al).

[0015] Gorbach analysed urinary isoflavone excretion in Japanesesubjects who consumed a traditional Japanese low-fat diet. The presenceof estrogenic isoflavones in the urine of the women, men and childrenstudied suggested to Gorbach that the isoflavones produced a therapeuticeffect. The obvious flaws in this study, namely that in a diet with highisoflavone intake significant urinary out-put of isoflavones would beexpected, and the huge number of biochemically active species in anydiet make it impossible to ascribe biological effects to any particularcomponent or components indicate that Gorbach's claims do not standscrutiny. The fact that a community with a particular health profilehappens to have a high dietary level (and high body levels) of a certainplant component in no way establishes cause and effect. This is onlyachieved through appropriately conducted clinical studies where wellaccepted scientific principles can be applied.

[0016] Clinical and other studies done to date in this area are highlyequivocal, with no consistent effect reported. Reported studies haveinvolved the challenge of peri-menopausal women either with wholefoodstuffs (such as soyflour) containing isoflavones or with extracts ofsoya or other legumes, often together with other agents such asvitamins, or isoflavones together with estrogen and/or vitamins andvarious minerals. It is to be noted that soy does not contain theisoflavones formononetin and biochanin. Even when a positive clinicaleffect has been obtained, it has been with a mixture of a plurality ofisoflavones, as well as a wide range of other unidentified dietarycomponents and other biologically active components—it is known forexample that other compounds present in legumes such as flavonoids (e.g.quercetin, luteolin, kaempferol and lignans) also are estrogenic and itis also likely that among the other 700 or so isoflavonoids present inthe Leguminosae family there are as yet unidentified isoflavonoids withestrogenic activity.

[0017] Gorbach suggests that isoflavanoids bind to estrogen receptors,exerting an estrogenic effect in menopausal women. However, it isacknowledged generally that no direct evidence exists to link thepresence of isoflavones with a therapeutic effect in treatment and/orprevention of acute menopausal syndrome symptoms. But even if such alink could be inferred from the current epidemiological and clinicalstudies, which it can not, the question remains what, if any,therapeutic effect is the result of a collective effect of theestrogenic isoflavones—daidzein, formononetin, biochanin, genistein. Allfour isoflavones are estrogenic, but they have quite differentestrogenic potencies. The relative estrogenicity of genistein, daidzein,formononetin, biochanin is 1.3, 0.09, 0.01, 0.07 respectively (relativeto 17β-estradiol 100). Hence formononetin and biochanin have negligibleestrogenic activity. On this basis, and given the relative proportionsof daidzein and genistein in the blood of Japanese maintaining a typicalJapanese diet, it might be inferred that genistein potentially is themost potent isoflavone as far as acute menopause syndrome symptoms areconcerned.

[0018] Estrogenic isoflavones have also been identified as possibletherapeutic compounds in the treatment and/or prevention ofosteoporosis. Asian populations consuming large amounts ofphytoestrogen-rich soybeans and vegetables appear to be protected to agreater extent than western populations from the problems associatedwith osteoporosis. These observations are by no means clear, and arecontradicted in a number of studies.

[0019] Fujita and Fukase (Proc. Soc. Exp. Biol. Med. 200(2) 149-5, 1992)indicates that in osteoporosis analysis between Japanese and USpopulations diet is not of particular significance, with bone mass beingvery similar in both populations. Instead they suggest the outcomes ofosteoporosis are more likely associated with lifestyle affecting muscledevelopment and motor control. Arjanandi et al (American Institute ofNutrition, p161-167, 1995) indicates that any protective effects of soyon bone is associated with soy protein. Hunt et al (Am. J. Clin. Nutr.,p517-523, 1989) indicate that there is no appreciable difference in bonedensity between elderly menopausal omnivores, and elderly menopausalvegetarians whose diet included isoflavone rich plant materials.

[0020] Published European Patent Application No. 0135172 (Takeda,published May 27, 1985) discloses methods for the treatment ofosteoporosis by administration of 7,4-dihydroxy isoflavone (daidzein)relying on its estrogenic activity. This finding is inconsistent withthe biological studies reported above. Moreover, Tobe et at (Biosci.Biotech. Biochem. 61(2) 370-371, 1997) show that daidzein stimulatesbone resorption, that is bone breakdown, and would be contra-indicatedfor treating osteoporosis, as it would worsen an existing condition, andpossibly pre-dispose a non affected person to osteoporosis.

[0021] Against the foregoing background, the present invention ispredicated upon our surprising finding that, within the framework ofwhat was conjectured regarding the treatment/prevention of menopausalsymptoms and osteoporosis, formononetin may be used to treat bothmenopausal symptoms and osteoporosis, and daidzein may be used in thetreatment of menopausal symptoms. Our findings indicate thatformononetin has pronounced clinical activity in the treatment and/orprevention of menopausal symptoms and in the treatment and/or preventionof osteoporosis as does daidzein in the treatment/prevention ofmenopausal symptoms. This is highly unexpected given the negligibleestrogenic effect of formononetin, the bone resorption activity ofdaidzein and the established view that formononetin was very rapidlymetabolised to daidzein in the gut.

SUMMARY OF THE INVENTION

[0022] There is provided in a first aspect of this invention a methodfor the treatment or prevention of menopausal symptoms or osteoporosiswherein there is administered to a subject in need of such treatment atherapeutically effective amount of the isoflavone formononetin, or amethod for the treatment or prevention of menopausal symptoms whereinthere is administered to a subject in need of such treatment atherapeutically effective amount of the isoflavone daidzein, theisoflavone being optionally administered with one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.

[0023] In another aspect of the invention there is provided apharmaceutical composition for the treatment or prevention of menopausalsymptoms or osteoporosis wherein the said composition comprises theisoflavone formononetin or a pharmaceutical composition for thetreatment or prevention of menopause wherein said composition comprisesthe isoflavone daidzein together with one or more pharmaceuticallyacceptable adjuvants, carriers and/or excipients.

[0024] In another aspect of the invention there is provided use of theisoflavone formononetin in the treatment or prevention of menopausalsymptoms or osteoporosis, or use of the isoflavone daidzein in thetreatment or prevention of menopausal symptoms, the isoflavone beingoptionally administered with one or more pharmaceutically acceptableadjuvants, carriers, and/or excipients.

[0025] In a further aspect of the invention there is provided an agentfor the treatment or prevention of menopausal symptoms or osteoporosis,or an agent for the treatment or prevention of menopausal symptoms whichcomprises daidzein optionally in association with one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.

DETAILED DESCRIPTION

[0026] Throughout this specification and the appended claims, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising” or “include” or “including”, will beunderstood to imply the inclusion of a sated element or integer or groupof elements or integers but not the exclusion of any other element orinteger or group of elements or integers.

[0027] The present invention provides in a first aspect a method for thetreatment or prevention of menopausal symptoms or osteoporosis whereinthere is administered to a subject in need of such treatment atherapeutically effective amount of the isoflavone formononetin, or amethod for the treatment or prevention of menopausal symptoms whereinthere is administered to a subject in need of such treatment atherapeutically effective amount of the isoflavone daidzein, theisoflavone being optionally administered with one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.

[0028] It is believed that the invention described represents asubstantial breakthrough in the field of treatment and/or prevention ofmenopausal symptoms and osteoporosis. The administration to subjects ofplant extracts containing a range of isoflavones may be unpleasant inthe sense that the extract may not be particularly palatable, and/or maycontain a host of ill-defined compounds which may affect disadvantageousbiological activity. Wilcox et al (British Medical Journal (1990) 301:905) have reported increases in vaginal cell proliferation amongstpost-menopausal women consuming soybean phytoestrogens for six weeks. Inaddition Markiewicz et al (J. Steroid Biochem. (1993) 45: 399) haveshown experimentally that the soy isoflavone genistein exhibited anestrogen effect on endometrial cancer cells, that is, potentiated cancercell growth in this cell type. Such reports raise questions about thesafely of comparatively high doses of genistein. The present inventionprovides the treatment or prevention of menopausal symptoms andosteoporosis without any side effects caused by uncharacterisedbiologically active plant materials (such as coumesterols), or otherdisadvantageous effects.

[0029] The menopausal symptoms which may be treated according to themethod of this invention are those described by Greene, J. G. and Cooke,D. J. (1980) British Journal of Psychiatry Volume 136, 486-491.Preferably the menopausal symptoms treated or prevented according to thepresent invention are hot sweats and night time sweats, moreparticularly hot sweats. Having said this, the method of the inventionis applicable to the treatment and/or prevention of other symptoms ofmenopause as previously described. The isoflavone formononetin is of theformula (I):

[0030] Although it was previously thought that formononetin was almostimmediately metabolised demethylated) to daidzein upon administration toa subject, the present inventors have found that formononetin persistsin the blood stream for a considerable the (having a half life ofgenerally about 20 hours).

[0031] The isoflavone daidzein is of the formula (II):

[0032] Formononetin or daidzein arm preferably administered to a subjectsubstantially unaccompanied by other isoflavones. By this is meant thatany composition or preparations may contain minor amounts of otherisoflavones, in the order of 10% (w/w) or less. Preferably theformononetin or daidzein represents at least 90% of isoflavone content,more preferably 95%, even more preferably 98% or more. Genistein, ifpresents is in amounts of about 5% or less, more preferably less than 1%(w/w) with regard to isoflavone content. It is recognised by regulatoryagencies that an isoflavone content in the order of 95 % of totalisoflavones represents effective purity.

[0033] In the treatment of menopausal symptoms formononetin may beadministered in combination with daidzein, for example from a ratio of1:10 to 10:1.

[0034] Daidzein metabolites may be used in place of daidzein in thevarious embodiments of this invention. These metabolites include equol,o-desmethylangolensin (ODMA), dehydroequol, 2-dehydro-ODMA,6-hydroxy-ODMA, dihydrodaidzein and tetra-hydrodaidzein (whichcollectively may be referred to herein as daidzein metabolites).Accordingly, in a further aspect this invention extends to a method forthe treatment or prevention of menopausal symptoms or osteoporosiswherein, there is administered, to a subject in need of such treatmentand/or prevention, formononetin or a daidzein metabolite, optionallyadministered with one or more pharmaceutically acceptable adjuvants,carriers and/or excipients. The formononetin or daidzein metabolites maybe administered in a form substantially unaccompanied by otherisoflavones.

[0035] Daidzein and/or formononetin compositions or preparations areadministered in an amount, and under a dosage regime which gives reliefto menopausal symptoms or osteoporosis. With regard to menopausalsymptoms this can be readily determined by the subject who is beingtreated, or by their physician. Generally, it is found that preventionor therapy of menopausal symptoms and osteoporosis results from dailyadministration of formononetin such as from one to six times in a 24hour period, as does the treatment or prevention of menopausal symptomswith daidzein, so as to give a daily dose of the isoflavone in an amountfrom about 5 mg to about 400 mg per day (this dosage range may bereferred to as the “effective amount”).

[0036] Formononetin and daidzein may be prepared by synthesising thecompounds by conventional chemical synthetic techniques as are wellknown in the art, or by purification from extracts of plants of thegenus Leguminosae, particularly from soy (such as from soy flour, soyhypocotyls) and clover (such as red clover, and subterranean clover)such as to form a formononetin or daidzein composition or preparation.

[0037] Compositions/preparations administered to subjects for thetreating and/or prevention of, or for reducing the predispositon to,menopausal symptoms or osteoporosis may comprise in addition to thespecific isoflavones previously mentioned formononetin optionallyadministered with one or more pharmaceutically acceptable adjuvants,carriers and/or excipients, so as to form a composition or preparation.Pharmaceutically acceptable adjuvants, carriers and/or excipients, andthe like, are well known in the art, for example as described in theHandbook of Pharmaceutical Excipients, second edition, AmericanPharmaceutical Association, 1994 (incorporated herein by reference).Daidzein or formononetin may be administered in the form of tablets,capsules, powders for reconstitution, syrups, foods (such as food bars,biscuits, snack foods and other standard food forms well known in theart) or in drink formulations. Drinks may contain flavouring, buffersand the like.

[0038] In the method of this invention calcium may be co-administered(that is before at the same time or after the isoflavones previouslymentioned), for example as a separate tablet, or as part of a suitabledosage form.

[0039] In a further aspect of this invention there is provided apharmaceutical composition for the treatment or prevention of menopausalsymptoms or osteoporosis wherein the said composition comprises theisoflavone formononetin or a pharmaceutical composition for thetreatment or prevention of menopause wherein said composition comprisesthe isoflavone daidzein, together with one or more pharmaceuticallyacceptable adjuvants, carriers and/or excipients. As mentioned above,pharmaceutically acceptable adjuvants, carriers and/or excipients arewell known in the art. Examples include compositions according to thepresent invention may include one or more pharmaceutically acceptablecarriers. The carriers are selected so as to be acceptable in the senseof being ingredients in the composition and must not be deleterious tothe patient. The carriers may be solid or a liquid, or both, and may beformulated with the extract as a unit-dose, for example a tablet, whichmay contain from 0.5% to 59% by weight of the active compound or up to100% by weight to the active compound. Compositions may be prepared byany of the well known techniques of pharmacy, for example admixing thecomponents, optionally including excipients, diluents (for examplewater) and auxiliaries as are well known in the pharmaceutical field.

[0040] The compositions of the invention include those suitable fororal, rectal, optical, buccal (for example sublingual), parental (forexample subcutaneous, intramuscular, intradermal and intravenous) andtransdermal administration. The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand the state of the patient.

[0041] Compositions suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the extract; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active isoflavone andone or more suitable carriers (which may contain one or more accessoryingredients as noted above). In general the compositions of theinvention are prepared by uniformly and intimately admixing theisoflavone with a liquid or finely divided solid carrier, or both, andthen, if necessary, shaping the resulting mixture. For example, a tabletmay be prepared by comprising or moulding a powder or granulescontaining the extract, optionally with one or more accessoryingredients. Compressed tables may be prepared by compressing in asuitable machine, the extras in the form of a powder or granulesoptionally mixed with a binder, lubricant, inert diluents, and/orsurface active/dispersing agent(s). Moulded tablets may be made bymoulding, in a suitable machine, the powdered compound moistened with aninert liquid binder.

[0042] Suitable carriers may be fillers, such as sugars, for examplelactose, saccharose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, and also binders, such as starch pastes using, for example,corn, wheat, rice or potato starch, gelatin, tragacanth, methylceulloseand/or polyvinylpyrrolidone, and, if desired, disintegrators, such asthe above-mentioned starches, also carboxymethyl starch, cross linkedpolyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such assodium alginate. Excipients may be flow conditioners and lubricants, forexample silicic acid, talc, stearic acid or salts thereof, such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragée coresare provided with suitable, optionally enteric, coatings, there beingused, inter alia, concentrated sugar solutions which may comprise gumarabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titaniumdioxide, or coating solutions in suitable organic solvents or solventmixtures, or, for the preparation of enteric coatings, solutions ofsuitable cellulose preparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Dyes or pigments may be added tothe tablets or dragée coatings, for example for identification purposesor to indicate different doses of active ingredients.

[0043] Other orally administrable pharmaceutical compositions aredry-filled capsules made, for example, of gelatin, and soft, sealedcapsules made of gelatin and a plasticiser, such as glycerol orsorbitol. The dry-filled capsules may comprise the extracts in the formof granules, for example in admixture with fillers, such as lactose,binders, such as starches, and/or glicants, such as talc or magnesiumstearate, and, where appropriate, stabilisers. In soft capsules, theextract is preferably dissolved or suspended in suitable liquids, suchas fatty oils, paraffin oil or liquid polyethylene glycols, to whichstabilisers may also be added.

[0044] Formulations suitable for buccal (sublingual) administrationinclude lozenges comprising the extracts in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

[0045] Formulations suitable for rectal administration are preferablypresented as unit dose suppositories. These may be prepared by admixingthe isoflavones with one or more conventional solid carriers, forexample cocoa butter, and then shaping the resulting mixture.

[0046] Compositions may include calcium or other active agents suggestedto provide some amelioration of osteoporosis or symptoms of menopause.

[0047] Pharmaceutical compositions generally comprise from about 5 mg toabout 400 mg of the isoflavone/s and may be administered one or moretimes per day.

[0048] In a further aspect of this invention there is provided use ofthe isoflavone formononetin in the treatment or prevention of menopausalsymptoms or osteoporosis, or use of the isoflavone daidzein in thetreatment or prevention of menopausal symptoms, the isoflavone beingoptionally administered with one or more pharmaceutically acceptableadjuvants, carriers, and/or excipients.

[0049] In another aspect of the invention there is provided use, offormononetin, for the manufacture of a medicament for the treatment orprevention of menopausal symptoms or osteoporosis, or use of daidzein orformononetin for the manufacture of a medicament for the treatment orprevention of osteoporosis. Generally, the isoflavone is provided in theform of a medicament in association with one or more pharmaceuticallyadjuvants, carriers and/or excipients. Daidzein and/or formononetin maybe conveniently blended as a dry powder with other components and formedinto appropriate dosage forms. Such medicaments generally comprise fromabout 5 mg to about 400 mg of isoflavone.

[0050] According to a still further aspect of the invention there isprovided an agent for the treatment or prevention of menopausal symptomsor osteoporosis, or an agent for the treatment or prevention ofmenopausal symptoms which comprises daidzein optionally in associationwith one or more pharmaceutically acceptable adjuvants, carriers and/orexcipients.

[0051] Daidzein and/or formononetin may be administered in the form of adietary product, as mentioned above, for example in a palatable foodcarrier such as a confectionary bar, biscuit, cereal or beverage.

[0052] The methods and compositions of the present invention do notinclude the use of estrogens, or components such as licorice,cholecalciferol and vitamin E. Estrogen administration has many sideeffects such as genital bleeding and hepatic disorders. Licorice hasvasoactive activity and may exacerbate menopause symptoms orosteoporosis- Cholecalciferol and vitamin E are also disadvantageous.

[0053] The foregoing description in relation to daidzein, applies to thedaidzein metabolites equol, o-desmethylangolensin (ODMA), dehydroequol,2-dehydro-ODMA, 6-hydroxy-ODMA, dihydrodaidzein and tetra-hydrodaidzein,as well as combinations of the above species.

[0054] Hence daidzein may be replaced by one or more of thesemetabolites. Daidzein or formononetin may also take the form ofaglycones, glycosides, malonyl or acetyl derivatives.

[0055] This invention will now be described with reference to thefollowing non-limiting examples.

EXAMPLE 1

[0056] Peri-menopausal women with symptoms of acute menopausal syndromeincluding at least an average of 3 hot flush episodes per day weretreated with a preparation containing a concentrated amount of daidzein,formononetin, genistein and biochanin prepared according to the methoddescribed in published Australian patent application 40523/93(incorporated herein by reference). The preparation contained 100 mgisoflavones containing biochanin and formononetin (in the ratio of1.8:1). Urine samples were collected from each woman at the start of thetest and then again at the end of the trial and the levels of particularisoflavones in the urine (as a marker of total body isoflavone ratios)then correlated with the degree of reduction in the incidence of hotflushes per day over the course of the study. Daidzein and genisteinwere detected in the urine of all test subjects but the levels werehighly variable, ranging from barely detectable to relatively high.Formononetin and biochanin levels ranged between undetectable andsignificant. Genistein levels showed no correlation with therapeuticresponse; daidzein levels correlated well with therapeutic responseindicating potential therapeutic utility of daidzein and formononetin.

EXAMPLE 2

[0057] Daidzein substantially free of other isoflavones was preparedfrom soy by the following method. 1 kg of defatted soyflour (readilyavailable from many commercial sources) was added to 10 L of watercontaining 50 g of glucan hydrolase enzyme (BioFeed Beta CT, NovoNordisk, Denmark). To this suspension 5 L of ethyl acetate is thenadded. This mixture is mixed vigorously using a high pressure pump for 3hours so that it forms an emulsion. This mixing ensures effectivecontact between the aqueous phase and the miscelles of ethyl acetate sothat the enzymatically hydrolysed aglucone forms of the isoflavones movefrom the aqueous to the organic solvent phase. The three phases(soyflour, aqueous, ethyl acetate) are separated by centrifugation in aswing bucket centrifuge at 2000 g for 30 minutes. The upper phasecomprising ethyl acetate is aspirated, 200 ml of water added, and thenplaced into a rotary evaporator at 75° C. under a weak vacuum. Uponremoval of the ethyl acetate, the residual aqueous phase containing theisoflavones is extracted once with 500 ml of hexane to remove oils andfats, and then twice with 500 ml of octanol which selectively removesgenistein. The aqueous phase then was taken to dryness overnight in anoven at 80° C. This material was shown by high pressure liquidchromatographic analysis to comprise 65% isoflavones comprising daidzein(95%): genistein(5%): formononetin(0%): biochanin(0%). This material wasmixed with standard carriers/excipients and tableted to give 500 mgtablets containing 25 mg daidzein. In this particular example, thedaidzein was tableted with equivalent (w/w) amounts of microcrystallinecellulose, calcium hydrogen phosphate, magnesium stearate and anhydrouscolloidal silica.

[0058] Formononetin substantially free of other isoflavones was preparedfrom clover by enzyme/solvent as above. Formononetin was recovered as apurity level greater than 95% by chromatographic analysis or by HPLC.

EXAMPLE 3

[0059] The dried end product of the first part of Example 2 above can beused to further concentrate genistein or daidzein with/without. 3 kg ofthis material is mixed with 1000 L of an organic solvent such asacetonc, chloroform or octanone, but preferably acetone for reasons ofsafety and cost. Each of these 3 solvents has been shown by theinventors to have high affinity for genistein but not daidzein. Themixture is stirred continuously at room temperature for between 1-24hours but preferably 2 hours during which time a large amount(approximately 75%) of the genistein transfers into the solvent phase.The mixture is allowed to settle for about 2 hours, the solvent isseparated from the residue (Sample 2) and transferred to a still forevaporation. Sample 2 material preferably is extracted with acetone afurther 1-5 times (preferably 4 times). This material typically contains76% daidzein, 1% genistein, 0.5% glycetein, with the remaindercomprising residual lipid soluble material such as short chain fattyacids. On a w/w basis compared with the other isoflavones, thispreparation contains 98% (w/w) daidzein. Daidzein may be purified to 95%(w/w) of total material or more purity by preparative purificationregimens such as preparative HPLC. A daidzein preparation according tothis example is tableted with conventional inert excipients according toExample 3 to give a 200 mg tablet containing 50 mg daidzein.

EXAMPLE 4

[0060] A group of 36 post-menopausal women experiencing menopausalsymptoms were treated with a composition containing either 15 mgdaidzein or 60 mg daidzein administered on a daily basis for 3 months.Compared to a placebo control group there was a significant decrease inthe Greene Score for menopausal symptoms which corresponds totreatment/amelioration of menopausal symptoms (such as hot flushes). Theurinary profile of these subjects demonstrated the therapeuticeffectiveness of daidzein as against other isoflavones.

[0061] In a similar study the same dosage levels of formononetin againgave a significant decrease in Greene Score for menopausal symptoms asfor daidzein.

EXAMPLE 5

[0062] A pharmacokinetic study involving 16 human patients aged between18 and 40 was conducted, to determine the pharmacokinetics offormononetin following oral administration. Each patient was orallyadministered 9.3 mg of formononetin with 200 ml of purified water andmaintained a low isoflavone diet for one week prior to, and during thestudy. Blood samples were taken at 0, 0.25, 0.5, 0.75, 1.0. 1.5, 2.0,2.5, 3, 4, 5, 6. 8, 10 and 12 and 24 hours post administration, andanalysed for formononetin concentration.

[0063] An analysis of formononetin concentrations (ng/ml) against timedemonstrated that contrary to previous opinion, unmetabolisedformononetin persists in the blood stream for considerable timefollowing administration (having a half life of about 20 hours).

EXAMPLE 6

[0064] 15 post-menopausal women who are experiencing menopausal systemsare each administered 60 mg formononetin daily for three months.Relative to a control group the treatment group show a significantdecrease in Greene Score for menopausal symptoms.

[0065] A similar study involving 10 post-menopausal women in a high riskgroup for osteoporosis were also administered 60 mg formononetin dailyfor three months. Preliminary results indicate protection againstosteoporosis in the treatment group when measured by bone density andbone turnover markers were measured.

EXAMPLE 7

[0066] The second study (double-blind, placebo-controlled) involved asix month study of the effects of phytoestrogen formononetin on boneresorption markers in twenty post-menopausal women. The aim of the studywas to evaluate the efficacy of a defined daily quantity of isoflavoneat 25 mg, 50 mg or 75 mg on bone resorption makers in post menopausalwomen and compare these to normal controls The effects of isoflavones onendometrial thickness, circulatory lipids and coagulation factors werealso evaluated. Subjects were also given supplemental calcium in a doseof 1200 mg per day.

[0067] Bone density measurements using a bone densitometer were made atthree sites of the forearm at 0.3 and 6 months and showed significantimprovement in bone density. Bone markers for osteocalcin, deoxypyridinoline crosslinks, N-terminal collagen crosslinks, calcium andother markers showed a similar improvement in bone resorption andturnover.

[0068] It is to be recognized that the present invention has beendescribed by way of example only, and that various modifications and/oralterations which would be obvious to a person skilled in the art, onthe basis of the teaching herein, can be made thereto without departingfrom the intended scope or spirit of the invention.

1. A method for the treatment or prevention of menopausal symptoms orosteoporosis wherein there is administered to a subject in need of suchtreatment a therapeutically effective amount of the isoflavoneformononetin, or a method for the treatment or prevention of menopausalsymptoms wherein there is administered to a subject in need of suchtreatment a therapeutically effective amount of the isoflavone daidzein,the isoflavone being optionally administered with one or morepharmaceutically acceptable adjuvants, carriers and/or excipients.
 2. Amethod according to claim 1 for the treatment or prevention ofosteoporosis.
 3. A method according to claim 2 wherein formononetin isadministered to a subject.
 4. A method according to claim 1 for thetreatment or prevention of menopausal symptoms.
 5. A method according toclaim 4 wherein formononetin or daidzein is administered to a patient.6. A method according to claim 1, wherein the isoflavone is present inan amount of at least 90% (w/w) as against any other isoflavones.
 7. Amethod according to claim 1, wherein the isoflavone is administered atleast once per day.
 8. A method according to claim 1, wherein theisoflavone is administered in an amount from about 5 mg/day through toabout 400 mg/day.
 9. A method according to claim 1, wherein theisoflavone is administered from 1 to 6 times in a 24 hour period so asto give a daily dosage of from about 5 to about 400 mg.
 10. A methodaccording to claim 1 wherein maid isoflavone is derived from a plantextract comprising daidzein and/or formononetin substantiallyunaccompanied by other isoflavones.
 11. A method according to claim 1for the treatment or prevention of osteoporosis wherein a combination offormononetin and daidzein is administered to a patient.
 12. A methodaccording to claim 1 wherein the isoflavone is substantiallyunaccompanied by other isoflavones.
 13. A method according to claim 1wherein daidzein is replaced by a daidzein metabolite selected fromequol, o-desmethylangolensin (ODMA), dehydroequol, 2-dehydro ODMA,6-hydroxy-ODMA, dihydrodaidzein and tetra-hydrodaidzein.
 14. Apharmaceutical composition for the treatment or prevention of menopausalsymptoms or osteoporosis where the said composition comprises theisoflavone formononetin or a pharmaceutical composition for thetreatment or prevention of menopause wherein said composition comprisesthe isoflavone daidzein, together with one or more pharmaceuticallyacceptable adjuvants, carriers and/or excipients.
 15. A compositionaccording to claim 14, which is a composition for the treatment orprevention of osteoporosis.
 16. A composition according to claim 15which includes the isoflavone formononetin.
 17. A composition accordingto claim 14 which is a composition for the treatment and/or preventionof acute menopausal symptoms.
 18. A composition according to claim 17wherein the isoflavone is daidzein or formononetin.
 19. A compositionaccording to claim 17 which includes a combination of daidzein andformononetin.
 20. A composition according to claim 14 which comprisesfrom 5 to 400 mg of isoflavone.
 21. A composition according to claim 14wherein daidzein is replaced by a daidzein metabolite selected fromequol, o-desmethylangolensin (ODMA), dehydroequol, 2-dehydro-ODMA,6-hydroxy-ODMA, dihydrodaidzein and tetra-hydrodaidzein.
 22. Use of theisoflavone formononetin in the treatment or prevention of menopausalsymptoms or osteoporosis, or use of the isoflavone daidzein in thetreatment or prevention of menopausal symptoms, the isoflavone beingoptionally administered with one or more pharmaceutically acceptableadjuvants, carriers, and/or excipients.
 23. Use according to claim 22,wherein the isoflavone is administered in an amount from about 5 mg toabout 400 mg.
 24. Use according to claim 22 for the treatment orprevention of osteoporosis wherein the isoflavone is formononetin. 25.Use according to claim 22 for the treatment or menopausal symptomswherein the isoflavone is formononetin or daidzein.
 26. Use according toclaim 25 wherein a combination of daidzein and formononetin areadministered to a subject.
 27. Use of formononetin, for the manufactureof a medicament for the treatment or prevention of menopausal symptomsor osteoporosis, or use of daidzein or formononetin for the manufactureof a medicament for the treatment or prevention of osteoporosis.
 28. Useaccording to claim 22 wherein the daidzein or formononetin isadministered from 1 to 6 times in a 24 hour period so as to give a dailydosage of about 5 mg to about 400 mg.
 29. Use according to claim 27wherein the isoflavone daidzein is combined with formononetin.
 30. Anagent for the treatment or prevention of menopausal symptoms orosteoporosis which comprises formononetin, or an agent for the treatmentor prevention of menopausal symptoms which comprises daidzein,optionally in association with one or more pharmaceutically acceptableadjuvants, carriers and/or excipients.
 31. A method or compositionaccording to claim 1 or 14, wherein the isoflavone daidzein is in theaglycone, glycoside, malonyl or acetyl form.